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Section: Cellular molecular
Senior Editor: Dr Larry Trussell
Oxytocin-induced postinhibitory rebound firing facilitates
bursting activity in oxytocin neurons
Jean-Marc Israel1,2*, Dominique A. Poulain1,2 and Stéphane H.R. Oliet1,2
1Neuroscience
2
Research Center, Inserm U862, Bordeaux, France;
Université Victor Segalen Bordeaux 2, Bordeaux, France
Abbreviated title: GABAergic activity facilitates bursting firing
*To whom correspondence should be addressed at:
Inserm U.862, 146 rue Léo-Saignat, 33077 Bordeaux, France.
Phone:
+33
5
5757
3736
/
Fax:
+33
5
5757
3750
/
E-mail:
jean-
marc.israel@bordeaux.inserm.fr
Number of figures: 10
Number of pages: 30
Number of words for abstract: 234
Number of words for introduction: 433
Number of words for discussion: 1232
Keywords: Hypothalamus, supraoptic, GABA, calcium current, neuroendocrine, lactation
Acknowledgements: We thank Dr. D.T. Theodosis and Pr. D. Voisin for critical
reading of the manuscript and N. Dupuy for technical assistance with the cultures.
ABSTRACT
During parturition and lactation, neurosecretory oxytocin (OT) neurons in the
hypothalamus achieve pulsatile hormone secretion by coordinated bursts of firing that
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occur throughout the neuronal population. This activity is partly controlled by
somatodendritic release of OT which facilitates the onset and recurrence of synchronized
bursting. To further investigate the cellular mechanisms underlying the control exerted by
OT on the activity of its own neurons, we studied the effects of the peptide on membrane
potential and synaptic activity in OT neurons in hypothalamic organotypic slice cultures.
Bath application of low concentrations of OT (<100 nM) facilitated GABA-A receptormediated inhibitory transmission through a presynaptic mechanism without affecting
membrane potential and excitatory glutamatergic synaptic activity. The facilitatory action of
OT on GABAergic transmission was dose-dependent, starting at 25 nM and disappearing
at concentrations above 100 nM. As previously shown, higher concentrations of OT (>500
nM) had the opposite effect, inhibiting GABA-A receptors via a postsynaptic mechanism.
Surprisingly, OT-mediated facilitation of GABAergic transmission promoted action potential
firing in 40% of the neurons. Each action potential occurred at the end of the repolarizing
phase of an inhibitory potential. Pharmacological dissection revealed that this firing
involved the activation of low-threshold activated calcium channels. Detailed statistical
analysis showed that OT-mediated firing up-regulated bursting activity in OT neurons. It is
thus likely to optimize OT secretion and, as a consequence, facilitate delivery and milk
ejection in mammals.
INTRODUCTION
Oxytocin (OT) is a hormone synthesized in magnocellular neurons that are located in
the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. During
parturition and lactation in the rat, OT neurons display periodic high frequency bursts of
action potentials (AP) that are synchronized in the whole OT neuron population. This
triggers a massive and pulsatile release of OT in the blood stream which, in turn, promotes
pup delivery and milk ejection (Poulain and Wakerley,1982; Wakerley et al.,1988). OT is
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also released from the somatodendritic compartment, a process that enables OT neurons
to regulate their own activity (Richard et al., 1991). Locally released OT is essential to the
onset of the milk ejection reflex (Moos et al., 1989; Neumann et al., 1993) and it enhances
the amplitude and frequency of suckling-induced bursts, an effect mimicked by injections
of OT in the 3rd ventricle (Freund-Mercier and Richard,1984). Conversely, injection of an
OT receptor (OT-R) antagonist directly in the SON or PVN greatly reduces bursting activity
(Lambert et al., 1993). Furthermore, OT appears to have the property of priming and
inducing its own release, thereby amplifying its local and long distance action (Moos et al.,
1984; Ludwig and Leng, 2006).
In vitro recordings have revealed that the periodic high frequency bursting of OT
neurons was driven by OT-sensitive glutamatergic inputs (Jourdain et al., 1998; Israel et
al., 2003). In addition, OT at fairly high concentrations (1-10 µM) is known to inhibit
glutamate (Kombian et al., 1997) and GABA release (De Kock et al., 2003) through
presynaptic mechanisms as well as GABA-A receptor-mediated responses via a
postsynaptic process (Brussaard et al.,1996). How these different effects come into play to
modulate OT neuron excitability, especially during lactation, remains unknown. To address
this issue we investigated the effects of OT applied at very low to large concentration (25
to 2000 nM) on identified OT neurons in vitro (Jourdain et al., 1996). Low concentration of
OT (<100 nM) did not affect membrane potential and excitatory postsynaptic activity but
triggered or accelerated GABA-A receptor mediated synaptic responses, through a
presynaptic action. In about 40% of OT neurons, the enhancement of inhibitory
transmission had the unexpected consequence of facilitating AP firing through a
postinhibitory rebound (PIR) following individual inhibitory postsynaptic potentials and
involving the activation of a low voltage-activated calcium current, as revealed by
pharmacological analysis. We found that OT-mediated PIR firing was responsible for the
increase in firing rate observed in OT neurons prior to burst occurrence, an increase tightly
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correlated to burst amplitude, thus providing a further mechanism for optimizing hormone
output during pup delivery and lactation in mammals.
MATERIAL AND METHODS
Slice preparation
Cultured slices were prepared using the roller tube method, as described previously
(Jourdain et al., 1996). Briefly, 4- to 6-day-old female Wistar rats were anaesthetized with
isoflurane (95% O2 and 5 % isoflurane) for 1 minute and decapitated. Brains were
removed, and tissue blocks that included the hypothalamus were quickly dissected and
sectioned (400 µm). Frontal slices containing the supraoptic nucleus (SON) were cut into
two parts along the third ventricle, and each part was placed on a glass coverslip coated
with heparinized chicken plasma. Thrombin was then added to the coverslip to coagulate
the plasma and permit adhesion of the slice to the coverslip. The coverslip was inserted
into a plastic flat-bottomed tube (Nunc, Roskilde, Denmark) containing 750 µl of medium
(pH 7.4; 295 mosmol/kg), composed of 50% Eagle’s basal medium (Life Technologies,
Gaithersburg, MD), 25% heat-inactivated horse serum (Life Technologies), and 25%
HBSS (Life Technologies) enriched with glucose (7.5 mg /ml) and 2 mM L-glutamate
(Seromed, Berlin, Germany). No antibiotics were used. The tubes were tightly capped and
inserted in a roller drum; the tubes were rotated approximately 15 turns/hr. The medium
was replaced twice a week.
Recordings were performed in 2-10 week-old cultures using a temperature-controlled
chamber (36.0±0.2 °C) perifused with a solution containing (in mM): NaCl 125; KCl 3;
MgSO4 1; KH2PO4 1.25; NaHCO3 5; CaCl2 2; glucose 5; HEPES 10 (pH 7.25; 290-295
mosmol/kg). Intracellular microelectrodes were filled with 1 M potassium acetate and 1 %
biocytin (Sigma). Electrode resistance varied from 150 to 250 Mohms. The patch clamp
technique was used in whole cell configuration (current or voltage clamp mode) using
electrodes (4-8 Mohms) filled with a solution containing (in mM) 120 K-gluconate, 20 KCl,
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10 HEPES, 1 EGTA, 1.3 MgCl2, 0.1 CaCl2, 2 Mg-ATP and 0.3 GTP. For IPSC recording,
electrodes were filled with (in mM): 141 CsCl, 10 HEPES, 5 QX-314-Cl and 2 Mg-ATP.
Series resistance (10-25 Mohms) was monitored on line and cells were excluded if more
than 20% change occurred during the experiment.
Signals were filtered at 2 kHz, digitized at 5 kHz and analyzed using pClamp 9
(Molecular Device, USA). Firing rate preceding high frequency burst was estimated from
frequency histograms calculated over 0.5 s integration periods and plotted versus time
using pClamp9. Pre-burst period was defined as the period occurring 20 s before burst
incidence. A change in basal firing frequency was considered as significantly different
when changes exceeded 10% of control values measured during the 200 s period
preceding the pre-burst period (Gouzènes et al, 1998). Detection of synaptic events was
achieved off-line using a sliding template whereas action potentials were detected using
an amplitude threshold (AxoGraph Scientific, USA). An action potential was considered
triggered by an IPSP if occurring within 300 ms of IPSP onset. Values are expressed as
means ± SD. Data obtained were compared statistically with the nonparametric
Kolmogorov-Smirnov test or the paired Student’s test.
Drugs
The following were added to the bath medium when required: synthetic OT
(Peninsula), the OT-R agonist, [4-threonine, 7-glycine]-oxytocin ([Thr4, Gly7]-OT ([4-7]
OT), the OT-R antagonist, desGly-NH2d(CH2)5[-DTyr2,Thr4]OVT (d-OVT; gifts from Dr.
Manning),
6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX,
RBI),
D(-)-2-amino-5-
phosphonopentanoic acid (AP5), ZD 7288 (Tocris), bicuculline, CsCl, picrotoxin and
tetrodotoxin, mibefradil (Sigma). GABA (Sigma) was dissolved in normal medium at 0.5
mM and was locally delivered through a micropipette (1-2 µm in diameter) positioned at
50-100 µm from the tested cell and connected to a pneumatic ejection system
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(Picospritzer, Intracel Ltd, UK).
Identification of recorded neurons
At the end of the recording, neurons were filled with biocytin (1%) using
hyperpolarizing current pulses. This was not necessary for patch clamp recording. Slices
were then fixed in 4% paraformaldehyde and 0.15% picric acid for 2 h at room temperature
and rinsed in 4% paraformaldehyde (2 x 20 min). Biocytin was visualized with streptavidinconjugated Texas Red fluorescence (Biosys, Compiègne, France) with appropriate filters
(Leitz DMR microscope, Leica, Paris). Slices then underwent double immunofluorescence
for OT or vasopressin, using a mixture of primary antibodies, one being a monoclonal
mouse immunoglobulin (Ig) raised against OT-related neurophysin (OT-NP, provided by
Dr. H. Gainer), the other, a polyclonal rabbit serum raised against vasopressin-associated
neurophysin (VP-Np, provided by Dr. A. Robinson).
RESULTS
All the results reported in this study have been obtained in 153 OT magnocellular
neurons which were identified according to two criteria: i) their ability to display high
frequency bursts of action potentials which are a specific property of OT neurons (Jourdain
et al, 1998; Israel et al, 2003), and ii) post-hoc immuno-identification. Intracellular
recordings (n=122) obtained from these neurons revealed a mean resting membrane
potential of 54.6±5.0 mV (n=50), a mean input resistance of 237.0±40.9 Mohms (n=50)
and action potentials (APs) of 71.9±10.9 mV (n=250 from 50 cells).
OT up-regulates GABAergic transmission
Intracellular recordings in current clamp mode in the presence of TTX (1µM),
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bicuculline (15 µM), CNQX (10 µM) and APV (40 µM) indicated that bath-applications of
OT (10 to 1000 nM) did not affect the resting membrane potential nor the input resistance
of OT neurons (Fig. 1A1-A3). Bath-application of OT (100 nM) in normal medium did not
alter the amplitude (98.4±5.2 % of control; n=8, P>0.05) or frequency (95.4±3.6 % of
control, n=8, P>0.05) of excitatory postsynaptic potentials (EPSPs) recorded at -80 mV
(data not shown). Under conditions where EPSPs were blocked with CNQX (10µM) and
AP5 (40 µM), low concentrations (25 nM to 100 nM) of OT significantly and reversibly
increased inhibitory postsynaptic potentials (IPSPs; Fig.1B) and currents (IPSCs; Fig. 2A)
in 56 out of 78 neurons (72%). In the remaining neurons (28%), OT did not affect
GABAergic activity (Fig. 1B) and these cells were thus considered as non-responsive to
the neurohypophysial peptide. In OT-responsive cells, the enhanced IPSP activity was
associated with an increase in the frequency (257±41 % of control; P<0.05; n=7) and
amplitude (211±52 % of control; P<0.05; n=7) of spontaneous events (Fig. 2B and 2C).
OT-sensitive IPSPs/IPSCs were blocked by bicuculline (15 µM), showing that synaptic
events modulated by OT were mediated by GABA-A receptors (not shown). The effect of
OT was mimicked by a specific OT-R agonist, [4-7] OT (100 nM; n=6) which increased the
frequency (377±82 % of control; P< 0.05) and amplitude (283±78 % of control; P<0.05) of
spontaneous IPSPs (Fig. 2C). Conversely, the effect of 50 nM OT was blocked in the
presence of 1 µM d-OVT, a specific OT-R antagonist (79±31 % and 91±9 % of control in
frequency and amplitude, respectively; n=4; Fig. 2C).
To identify the locus of action of OT, TTX (1 µM) was added to the external solution
to block AP-driven inhibitory synaptic events and thus make sure that monoquantal
synaptic responses (miniatures) were recorded. For these experiments we used the
whole-cell patch clamp technique in voltage-clamp configuration instead of intracellular
sharp electrode recording. Under these conditions, OT (50 nM) significantly increased the
frequency (198.3±29.3 % of control, n=4; P<0.05) but not the amplitude (103.5± 8.1 % of
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control, n=4; P>0.05) of miniature IPSCs (Fig. 2D-F). Although this set of data implies that
OT-R are located presynaptically on GABAergic neuron terminals impinging upon OT
cells, they do not rule out the possibility that OT-R are also located on GABA neuron
somata or on other neurons contacting GABAergic cells, and that these receptors also
contribute to facilitate inhibitory activity in OT neurons.
Bimodal dose-dependent action of OT on inhibitory transmission
The stimulatory effect of OT on GABAergic transmission was dose-dependent, with
a threshold of 25 nM and a maximal facilitation at 50 nM (Fig. 3A). Higher concentrations
of OT progressively inhibited GABAergic synaptic activity. The inhibition was almost total
with 300 nM OT, a result in agreement with the postsynaptic inhibitory action of OT on
GABA-A receptors previously reported in the SON (Brussaard et al., 1996). The dynamics
and dose-dependency of these two opposite effects of OT on GABAergic transmission
were then compared by monitoring simultaneously synaptic currents and responses
obtained with local puffs of GABA. Whereas low concentrations of OT (50 nM) triggered
IPSP activity without affecting the amplitude of GABA-induced responses, IPSP amplitude
and frequency gradually decreased when increasing OT concentrations, with a complete
inhibition obtained at 1000 nM (Fig. 3B and 3D). In the same recordings, GABA-induced
responses were slightly affected when OT concentration reached 500 nM and were
completely inhibited with 2000 nM (Fig. 3C and 3D). These findings demonstrate that OT
acts both at pre- and postsynaptic levels, depending on its concentration, to up- or downregulate GABAergic transmission.
OT-mediated IPSPs facilitate firing
In intracellular current-clamp recordings, low concentrations of OT (50-100 nM)
increased the firing activity in 11 out of 27 OT neurons (183±17 % of control; Fig. 4A1-A2).
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To identify the cellular mechanism responsible for this increase in firing rate, and its
possible relation to OT-mediated facilitation of inhibitory transmission, we investigated the
action of OT on OT neuron electrical activity in the presence of CNQX to block EPSPs
(Fig. 4B). Whereas CNQX inhibited AP firing, as previously reported (Jourdain et al.,
1996), OT still triggered spiking activity at resting membrane potential in 15 out of 36
neurons (41%). Examination of recordings at high resolution revealed that during such OTtriggered activity, most APs occurred at the end of the repolarizing phase of individual
IPSPs (Fig. 4B3-B4). This was particularly clear during OT washout where IPSP frequency
decreased to values < 5 Hz making it easier to reveal the link between IPSP and AP firing
(Fig. 4B3d). That APs were exclusively governed by IPSPs was confirmed in a series of
experiments where this OT-triggered firing occurring in the presence of CNQX was
completely inhibited by the specific GABA-A receptor antagonist picrotoxin (5 µM; Fig. 5A;
n=5). It is worth noting that we never observed an increased in AP firing without an
increase in IPSP activity.
The interaction between OT-mediated IPSP activity and the facilitation of firing
activity was confirmed in another set of experiments in which APs were triggered in
response to membrane depolarization in the presence of ionotropic glutamate receptors
inhibitors (CNQX and AP5). As illustrated in figure 5B1, OT neurons started to fire APs
only once the membrane potential reached spike threshold (-38.1±4.3 mV, n=7). In the
same recordings, OT triggered spike discharge without depolarizing the membrane
potential (Fig. 5B2), an effect that was accompanied by a consistent increase in IPSP
activity preceding the appearance of APs. These findings suggest that OT, by triggering or
dramatically increasing the occurrence of IPSPs, paradoxically facilitates firing activity in
OT neurons. These results prompted us to investigate the cellular mechanism underlying
this phenomenon.
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Ionic mechanisms underlying OT-induced firing
In presence of CNQX, each OT-triggered spike occurred at the end of the
repolarizing phase of an individual IPSP (Fig. 4B4). Such process is reminiscent of
postinhibitory rebound (PIR), as described in other structures (Angstadt et al., 2005;
Bertrand and Cazalets, 1998 ; Sohal et al., 2006). PIR is defined as the depolarization that
occurs at the offset of a hyperpolarizing period. At least, two non-exclusive mechanisms
might account for PIR-induced spikes. One involves low voltage-activated (LVA) Ca2+
channels that are first de-inactivated by hyperpolarization and then activated upon the
repolarization period (Bertrand and Cazalets, 1998, Jahnsen and Llinas, 1984; Fan et al.,
2000), thereby generating a depolarization known as a low threshold spike (LTS). This
LTS, if of a sufficient amplitude, can generate APs (Huguenard, 1996). A second
possibility is the activation of a hyperpolarization-activated inward current (IH) which
underlies rebound responses in many neurons (Matsushima, 1993; Straub et al., 2001;
Sekirnjak and du Lac, 2002).
Both LVA Ca2+ current and IH have been described in SON and PVN neurons (Fisher
and Bourque, 1995; Ghamari-Langroudi and Bourque, 2000; Luther and Tasker, 2000).
Thus, we first checked for their presence in our cultured slices before studying their
respective contribution to OT-mediated PIR-firing. Since the presence of LVA Ca2+
channels is associated with the generation of a LTS, we applied brief (50 ms)
hyperpolarizing pulses in the presence of TTX to block Na+-dependent APs. As illustrated
in figure 6A1, such pulses triggered a rebound depolarization typical of a LTS in 17 out of
32 neurons, a process that was compromised when the amplitude of the negative step
was reduced, as previously described (Erickson et al., 1993). In the absence of TTX, such
hyperpolarizing pulses triggered rebound APs (Fig. 6A2). To study the contribution of LVA
Ca2+ channels to this process, we bath-applied Ni2+ at 100 µM, a concentration that inhibits
T-type Ca2+ currents (Fisher and Bourque, 1995) and blocks LTS (Erickson et al., 1993) in
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SON neurons. In agreement with a role for these channels in LTS generation, APs
triggered by long (>100 ms; n= 5; Fig 6B1 and 7B1) or brief (25 ms; n=6; Fig 6B2 and 7B2)
hyperpolarizing pulses were completely abolished in the presence of this inhibitor.
Because Ni2+ might also interact with other voltage-gated Ca2+ channels, we tested the
action of mibefradil, a compound considered to be a specific T-type channel antagonist
(Van der Vring et al, 1999). At a concentration of 40 µM, mibefradil also inhibited pulsetriggered APs (n=5; Fig. 6B3 and 7B1). Taken together, these data suggest that the LVA
Ca2+ channels mediating LTS in these neurons are of the T-type.
We then examined the role of IH in this process. During 100 ms long hyperpolarizing
pulses, we reliably observed the typical depolarizing sag in the voltage response (e.g. Fig.
6C1 and 6D1) that reflects activation of IH (Ghamari-Langroudi and Bourque, 2000). As
previously reported (Ghamari-Langroudi and Bourque, 2001), this sag was inhibited by 3
mM external Cs+ (n=4; Fig. 6C1) or by 50 µM ZD 7288 (ZD; n=5; Fig. 6D1), two wellknown blockers of IH. Conversely, this sag was not affected in the presence of Ni2+ or
mibefradil (Fig. 6B1 and 6B3). Interestingly, blockade of I H with Cs or ZD did not prevent
pulse-triggered rebound spikes (Fig. 6C1, 6D1 and 7B1) even when hyperpolarization was
adjusted to that obtained in control conditions to compensate for changes in membrane
resistance (Fig. 6C2 and 6D2). The lack of effect of ZD on pulse-triggered APs was also
observed with pulses of shorter duration (25 ms; n=6; Fig 6D3 and 7B2).
Ni2+, mibefradil, Cs+ and ZD were then used to assess the respective contribution of
LVA Ca2+ channels and IH to OT-mediated PIR firing. As illustrated in figure 7A and
summarized in figure 7C, Ni2+ (n=4) and mibefradil (n=5) inhibited AP firing, but not IPSP
activity (Fig. 7D), triggered by OT. On the other hand, neither Cs + (n=4) nor ZD (n=4)
affected significantly OT-triggered firing activity (Fig. 7A and 7C) or IPSPs (Fig. 7D). These
data suggest that IPSPs can trigger rebound firing through the recruitment of LVA Ca 2+
channels. If this is true, a rebound depolarization should be observed following individual
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IPSPs. In 4 cells where OT-triggered AP firing and IPSP activity were not intense enough
to mask such a phenomenon, rebound excitations were clearly observed following
inhibitory synaptic potentials (Fig. 8A1 and 8A2; also see fig 4B3 trace d). These rebounds
were not affected by the subsequent application of ZD (Fig. 8B1 and 8B2) whereas they
were completely abolished in the presence of Ni2+ (Fig. 8C1 and 8C2). As illustrated on the
averaged traces in figure 8D and from cumulative histograms in figure 8E, inhibition of the
rebound depolarization with Ni2+ resulted in an increased IPSP width, an effect that was
not observed with ZD. We thus used IPSP duration to assess the effect of the different
blockers on IPSP-triggered postinhibitory rebound excitation. Whereas both Ni2+ and
mibefrendil increased IPSP duration (12612 % of control, n=4 for Ni2+; 1219 %, n=4, for
Mibefradil), neither ZD nor Cs+ modified significantly this parameter (Fig 8F). Taken
together, these findings reveal the involvement of LVA Ca2+ channels, but not of IH, in
IPSP-mediated postinhibitory rebound firing. These data also indicate that IPSPs have to
be of sufficient amplitude to trigger AP firing, which may not be the case under control
conditions.
Physiological relevance
Throughout this study, OT neurons recorded in the absence of glutamatergic and
GABAergic blockers usually displayed a bursting activity, either spontaneously or in
response to bath application of 100 nM OT (Fig. 9A; Jourdain et al., 1998, Israel et al.,
2003). This activity is characteristic of that recorded in vivo in lactating rats (Lincoln and
Wakerley,1975). Careful analysis of this activity in cultured slices revealed an increase in
firing rate occurring just before burst onset in 40% of the neurons (Fig. 9A). Such
increases in background firing rate immediately preceding the bursts have been already
reported in vivo where they are directly and positively correlated to the magnitude of the
bursting activity itself (Lincoln and Wakerley,1975). This prompted us to investigate
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whether a similar type of correlation prevailed in OT neurons recorded from organotypic
slice cultures, and to test whether OT-mediated PIR firing was playing a role in this
process.
Within bursts, both the mean AP frequency and the peak frequency (over 0.5 s) were
increased (175±38 % of control and 183±40 % of control, respectively, n=25) in neurons
showing an enhanced background firing activity prior to burst onset (Fig. 9B1-B2). To
further analyze burst magnitude, we used the same index as described by Lincoln and
Wakerley which corresponds to the number of spikes within the burst multiplied by the
peak frequency (Lincoln and Wakerley, 1975). As illustrated in figure 9C, the burst index
was positively correlated (r=0.61; n=48 bursts from 15 cells) to the firing frequency
measured 20 seconds before the incidence of each burst, a result in complete agreement
with previous in vivo data (Lincoln and Wakerley, 1975; Brown et al., 2000).
Although the bursts are driven by glutamatergic inputs (Jourdain et al., 1998; Israel et
al., 2003), the origin of the increase in background firing rate in OT cells is unknown. One
possibility is that this phenomenon is due to OT-mediated PIR firing. To test this
hypothesis, we analyzed synaptic activity just before the occurrence of each burst. EPSP
activity occurring during this period remained unchanged whether an increase in
background firing rate occurred or not (Fig. 10A). On the contrary, in neurons that
displayed an increase in firing rate prior to the bursts, a marked increase in both amplitude
(155±12 % of control; n=10; P<0.05) and frequency (189±39 % of control; n=10; P<0.05)
of IPSPs occurred (Fig. 10A-B). These findings strongly support a relationship between
IPSP activity and increased background firing rate. Because IPSP-mediated PIR firing is
related to OT, it is likely that the increased firing observed in these neurons resulted from
the dendritic release of endogenous OT. If this is true, then activation or inhibition of OT-R
should affect background firing and, consequently, the burst index. In agreement with this
hypothesis, OT (50-100 nM) increased the mean firing rate prior to burst onset from
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2.3±0.7 Hz to 4.78±1.24 Hz (211±41 % of control, n=5; Fig. 9C, 10C1 and 10D) whereas
the OT-R antagonist, d-OVT, decreased it from 2.98±0.20 Hz to 0.98±0.40 (32±14 % of
control, n=4; Fig. 9C, 10C2 and 10D). In these neurons, OT and d-OVT respectively
augmented (163±26 %, n=5) and diminished (36±19 % of control) the burst index as
expected (Fig. 10D). It is noteworthy that in the presence of OT, a concomitant increase in
IPSP frequency (190±26 % of control, n=5, P<0.05) and amplitude (191±35 % of control,
n=5, P<0.05) occurred (Fig. 10E) whereas d-OVT by itself, induced an opposite effect
(frequency: 73±6 % of control, n=4, P<0.05; amplitude: 56±9 % of control, n=4, P<0.05;
Fig. 10E). This suggests that endogenous ambient OT has a positive action on IPSP
activity and, consequently, on burst magnitude.
DISCUSSION
Action potential firing in neurons is usually obtained when the membrane potential is
depolarized above spike threshold. This generally occurs through activation of ion
channels or ligand-gated receptors or through the relief of tonic inhibition, a process
known as disinhibition. Another mechanism promoting neuronal firing, although less usual,
is postinhibitory rebound (PIR). In this phenomenon, one to several APs can be generated
during the membrane repolarization that follows the offset of a hyperpolarizing event. PIR
may involve hyperpolarization-activated current (IH), de-inactivation of voltage-gated Ca2+
currents or both. Such process is responsible for triggering activity in motoneurons
(Bertrand and Cazalets, 1998) in thalamocortical neurons (Sohal et al., 2006) and in rat
caudal hypothalamic neurons (Fan et al., 2000) for example. Here we described a process
in which a peptide, oxytocin, by facilitating the occurrence of hyperpolarizing GABAergic
synaptic potentials, promotes AP discharge through PIR firing. This process potentiates
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bursting activity of OT neurons which is responsible for the massive and intermittent
release of OT in the blood, and thus for pup delivery and milk ejection.
OT modulation of firing activity in OT neurons
The low concentrations of OT that we used here (25-100 nM) is more compatible with
physiological concentration, as suggested by microdialysis experiments (Neumann et al.,
1993). Interestingly, the facilitatory action of OT on IPSP activity was observed in 72% of
OT neurons. That such low concentrations of OT accelerate firing in OT-responsive
neurons through the generation of IPSPs is paradoxical since hyperpolarizing synaptic
potentials are usually associated with inhibition rather than facilitation of firing activity. The
action of OT was receptor-mediated since it was mimicked by an OT-R agonist and
inhibited by an OT-R antagonist. Similar up-regulation of GABAergic activity has been
reported in CA1 hippocampal neurons (Zaninetti and Raggenbass, 2000) and in putative
vasopressin hypothalamic neurons (Hermes et al., 2000) in response to OT and VP,
respectively. Furthermore, our experiments revealed that OT-mediated GABAergic
activation facilitated AP firing in about 40 % of OT neurons. Although moderate, this
increase in firing rate was in the range of that reported in lactating rat in vivo in response to
local OT applications (Brown et al., 2000).
How can a GABAergic inhibitory synapse become excitatory? Several mechanisms
may underlie IPSP-mediated PIR firing. One involves an LTS resulting from de-inactivation
of LVA Ca2+ currents, as previously described in the SON (Fisher and Bourque, 1995,
Erickson et al., 1993, Dudek et al., 1989). This is likely to be the case here for several
reasons. First, the percentage of neurons exhibiting an LTS is similar to that displaying
OT-triggered firing. Second, rebound OT-triggered depolarizations and spikes were
entirely blocked by Ni2+ at concentrations known to inhibit LTS and LVA Ca 2+ current in
these cells (Fisher and Bourque, 1995; Erickson et al., 1993) and by mibefradil, a more
specific T-type channel antagonist (Van der Vring et al, 1999). Although these data
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suggest that T-type channels are responsible for mediating IPSP-induced PIR firing in OT
neurons, the definitive demonstration for the implication of these channels awaits new and
more specific pharmacological tools. Similarly, it remains to be determined which CaVT
subunits among those already detected in hypothalamic neurons (Craig et al., 1999; Talley
et al., 1999), are implicated in this process. In SON neurons, activation of I H could also
account for PIR firing (Ghamari-Langroudi and Bourque, 2000): this can be ruled out since
Cs+ and ZD 7288 inhibited IH without affecting OT-mediated firing.
Heterogeneity of OT actions
It is clear from our observations that OT mediates distinct effects in the SON
according to its concentration. At 50 nM, OT stimulated the frequency and amplitude of
IPSPs/IPSCs, suggesting that it can act both pre- and post-synaptically. In the presence of
TTX, the frequency but not the amplitude of mIPSCs was increased, whereas postsynaptic
responses to applications of GABA were unaffected, indicating a presynaptic site of action
of OT. At concentrations higher than 50 nM, OT progressively inhibited both the frequency
and amplitude of IPSPs with a complete blockade obtained at 1000 nM, a result in
agreement with the inhibitory action of OT on GABA release previously described (De
Kock et al., 2003) and which has been related to the release of endocannabinoids acting
on presynaptic CB1 receptors (Oliet et al, 2007). At such high concentrations, OT also
inhibited GABA-A receptor mediated postsynaptic responses (Brussaard et al., 1996).
Taken together, our results reveal, therefore, that depending on its concentration, OT has
presynaptic effects, increasing then decreasing the probability of GABA release, and
postsynaptic effects, inhibiting GABA-A receptors on OT neurons.
It is obvious from this and our previous studies (Jourdain et al., 1998; Israel et al.,
2003) that OT acts differently, according to its concentration and its targets, namely,
GABA, glutamate and OT neurons. Such heterogeneity of actions may reflect differences
in OT-R mediating these responses. However, although there is evidence supporting the
17
existence of different receptor subtypes, only one type of OT-R has been described so far
(Gimpl and Fahrenholz, 2001). Alternatively, if there is only one type of OT-R, there may
be a differential expression of this receptor in different cells and/or different OT-R-coupled
second messenger pathways (Verbalis, 1999).
Physiological considerations
At parturition and during suckling, local release of OT from the somatodendritic
compartment is necessary to trigger and facilitate the periodic activation of OT neurons
(Freund-Mercier and Richard, 1984; Moos et al., 1984). We have shown previously that
OT neuron bursting is controlled by an intrahypothalamic network in which bursting
glutamate neurons govern OT neurons. In turn, OT somatodendritic release is essential to
modulate the bursting pattern of glutamatergic neurons (Jourdain et al., 1998; Israel et al.,
2003). The modulation of GABA transmission by OT as reported here may provide another
mean of generating APs during background activity, in addition to those generated by
EPSPs. Such a process may also explain previous in vivo data obtained in lactating rats
showing that locally applied GABA unexpectedly facilitated bursting activity (Moos, 1995)
whereas the same activity was impaired when GABA-A receptors were inhibited (Voisin et
al., 1995).
We here showed a strong correlation between background firing activity before each burst
and the magnitude of the bursts, a result similar to that reported in vivo (Lincoln and
Wakerley, 1975; Brown and Moos, 1997). One likely explanation to account for this
observation is that such an increase in firing rate facilitates the somatodendritic release of
OT, thereby increasing its ambient concentration and range of action in the extracellular
space. This, in turn, could positively modulate the intrahypothalamic pacemaker neurons
responsible for the bursting activity of OT-secreting cells (Jourdain et al., 1998). In
agreement with this hypothesis, we noticed that activation of OT-R with exogenous OT
increased background firing rate and, consequently burst magnitude, as reported in vivo
18
(Brown et al., 2000) whereas inhibiting OT-R with d-OVT had the opposite effect. In view
of these data, it appears that OT-mediated PIR firing is an important process by which OT
neurons could not only regulate their own activity but also influence the efficacy of the
intrahypothalamic network generating the bursting behavior responsible for pup delivery
and milk ejection. These results are reminiscent of those obtained in vivo where OT
neurons showing an increase in their background firing rate prior to the bursts have been
described as “leader” neurons whose task is to recruit “follower” neurons to optimize the
activation of the entire OT network, thereby maximizing synchronized bursting activity
(Moos et al, 2004).
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LEGENDS
Figure 1: OT facilitates inhibitory transmission.
A1: Example of a recording where OT was applied at two different concentrations (50
nM and 1 µM) in the presence of TTX, bicuculline, CNQX and AP5. The peptide did not
affect membrane potential nor membrane resistance measured from negative current
pulses of decreasing amplitude (from –150 to 0 pA by successive 15 pA steps). A2:
Voltage-current relationship obtained from the experiment shown in A1. The presence of
OT did not modify this relationship. A3: Histogram summarizing the lack of action of OT
(10 nM to 1 µM) on membrane potential (Em) and membrane resistance (Rm) in OT
sensitive-cells. The numbers of experiments are indicated in brackets. B: In the presence
of CNQX, application of 50 nM OT reversibly increased IPSP activity in 72 % of OT
neurons as illustrated in the example on the left panel (OT responsive cell). In the
remaining neurons (28%), GABAergic synaptic transmission was unaffected as shown on
the example illustrated in the right panel (OT non-responsive cell).
Figure 2: OT acts at presynaptic sites.
A: IPSC activity was increased in a reversible manner by 50 nM OT. B: Cumulative
distributions of IPSC intervals (left) and amplitude (right) indicated that OT increased both
the frequency and the amplitude of these events. C: Histogram summarizing the action of
26
OT, the agonist [4-7] OT and OT+ the antagonist d-OVT on IPSP frequency (Hz) and
amplitude (q). The number of cells is indicated in brackets. D: The activity of miniature
IPSCs recorded in the presence of TTX was also reversibly increased by OT (50 nM). E:
Cumulative distributions for miniature IPSC intervals and amplitude indicated that OT
increased the frequency without affecting the amplitude of these unitary events. F:
Summary histograms illustrating the effect of OT on the frequency and amplitude of
miniature IPSCs (n=4).
Figure 3: OT has bimodal effects on GABAergic transmission.
A: Histograms summarizing the changes in IPSP amplitude and frequency induced
by different concentrations of OT. Insets are example obtained from a cell where OT was
successively applied at 50 and 300 nM. OT triggered or facilitated IPSP activity at a
threshold concentration of 25 nM, an effect attenuated at higher concentrations (>100 nM).
Maximal facilitation for both IPSP amplitude and frequency was obtained at 50-100 nM
OT. B-C: Example of an OT neuron where OT, from 25 nM to 1000 nM, had distinct effects
on IPSPs (B) and on responses induced by local puffs of GABA (C). At 25 nM, OT slightly
increased synaptic activity without modifying the amplitude of the postsynaptic response.
With 50 nM OT, IPSP activity was dramatically augmented whereas GABA-induced
postsynaptic response was unaffected. At 500 nM, IPSP activity was strongly reduced
while the postsynaptic response was only slightly inhibited. Finally, 1000 nM OT inhibited
totally IPSPs and almost completely the postsynaptic response. D: Summary histogram
illustrating the action of OT at different concentrations on the amplitude of both IPSPs
(open bars) and GABA-induced responses (grey bars) obtained from the same neurons.
Number of experiments are indicated in brackets.
27
Figure 4: OT-induced IPSP activity triggers AP firing.
A1: Example of an OT neuron where exogenous application of OT (50 nM) increased
firing activity in a reversible manner as illustrated by the change in the sequential
frequency histogram. A2: Histogram summarizing the stimulatory effect of OT on firing
rate. The number of cells is indicated in brackets. B1: Under conditions where CNQX
totally abolished spontaneous firing (b), OT (50 nM) still triggered AP discharge (c). B2:
Summary histogram showing the stimulatory effect of OT in the presence of CNQX. B3:
Traces obtained from the recording shown in B1. In control conditions (a), APs were
mainly triggered by EPSPs. CNQX totally abolished EPSP activity and APs firing (b).
Subsequent addition of OT (50 nM) dramatically increased IPSP activity (arrow heads) and
restored AP firing (c). During washout of OT, although IPSP frequency and AP firing
decreased, spikes were still occurring at the end of IPSPs (d). B4: Magnification of the
trace c shown in B3 revealed that APs (*) occurred at the offset of IPSPs (arrow heads).
Figure 5: OT-triggered APs are exclusively governed by IPSPs.
A: In the presence of CNQX, OT (50 nM) dramatically increased IPSP activity and
firing (middle panel), an action that was totally blocked by picrotoxin (5 µM; right panel).
B1: In the presence of CNQX and AP5, a positive step current was required to depolarize
the cell and trigger firing. B2: OT (50 nM) under the same conditions and in the same
neuron, triggered IPSPs (arrowhead) and AP firing without depolarizing the membrane.
Figure 6: Presence of a low threshold spike (LTS) in OT neurons. A1: In the
presence of TTX, a negative current (50 ms, -40 pA, thick traces) injected in an OT neuron
at -55 mV, triggered a LTS (*) at the offset of the current step. This LTS was abolished
when the amplitude of the pulse was decreased (-30 pA, thin traces). A2: When negative
28
current steps (-50 and -70 pA, 40 ms) were applied from -55 mV, only the larger current
step triggered a rebound spike (thick trace). B: APs (thin traces) triggered by long (350 ms;
B1) and brief (25 ms; B2) hyperpolarized pulses were inhibited (thick traces) by 100 µM
Ni2+ (Ni). 40 µM mibefradil (Mib) also inhibited such pulses-triggered APs (B3). C1: A
depolarizing sag (*) typical of IH activation was observed during voltage responses to
negative current injections (thin traces). This sag was blocked with Cs + (Cs, 3 mM; thick
trace) whereas pulse-triggered APs remained unaffected. C2: In Cs+, adjusting the
hyperpolarization to the control value to compensate for the change in membrane
resistance associated with IH blockade did not affect pulse-triggered AP firing. D1:
Inhibition of IH with the specific antagonist ZD 7288 (ZD; 50 µM, thick trace), did not affect
200 ms long pulse-triggered APs. D2: As for Cs+, pulse-triggered AP firing was not
affected when the hyperpolarization was adjusted to the control value in the presence of
ZD. D3: APs triggered by brief (25 ms) hyperpolarizing pulses were not affected by ZD.
Figure 7: OT-mediated postinhibitory rebound firing is blocked by low threshold
activated calcium current blockers.
A: In CNQX-containing medium (control; left panel), firing activity triggered by 50 nM
OT (middle panel) was strongly inhibited by 100 µM Ni2+ (Ni) and 40 µM mibefradil (Mib)
whereas it was unaffected by 3 mM Cs+ (Cs) and 50 µM ZD 7288 (ZD). B: Histograms
summarizing the action of Ni2+ and ZD 7288 on APs triggered by long (>100 ms; B1) and
brief (25 ms; B2) hyperpolarizing pulses. C: Histograms summarizing the action of Ni 2+,
mibefradil, Cs+ and ZD 7288 on OT-induced PIR firing. D: Ni2+, mibefradil, Cs+ and ZD
7288 did not affect the frequency (Hz) nor the amplitude (q) of OT-triggered IPSPs.
Figure 8: IPSP-mediated rebound depolarization.
29
A1: Example of a recording obtained in the presence of CNQX and 50 nM OT showing the
presence of rebound depolarizations (*) occurring at the end of IPSPs. A2:
Superimposition of 8 consecutive IPSPs obtained from the recording in A1 clearly shows
that a rebound depolarization follows several of these inhibitory potentials. B1 and B2: In
the same recording, application of ZD 7288 (ZD) did not affect the occurrence of such
IPSP-triggered rebound depolarizations. C1 and C2: Subsequent addition of 100 µM Ni 2+
in the bathing solution completely abolished AP firing and rebound depolarizations. D:
Average IPSPs (n=93-105) obtained from the recordings shown in A, B and C. This graph
shows that rebound depolarization was unaffected by ZD (grey trace) whereas it was
abolished in the presence of Ni2+. Note that IPSP duration was increased with Ni2+. E:
Cumulative histograms representing the distribution of IPSP half-width obtained from the
recording shown in A, B and C. This distribution was significantly shifted toward higher
values in the presence of Ni2+. F: Summary histogram illustrating the percent change in the
half-width of OT-triggered IPSPs (n=4 cells). Whereas IPSP duration was significantly
increased by Ni2+ and mibefradil (Mib), it remained unchanged in the presence of Cs+ or
ZD.
Figure 9: Pre-burst firing and burst magnitude in OT neurons
A: Frequency histogram (FH) illustrating the bursting activity of an OT neuron
recorded in cultured slices. Insets show the raw recording at the time indicated before and
during a burst. B1: Superimposition of FHs recorded from 2 OT neurons displaying (grey)
or not (black) an increased firing activity prior to the occurrence of a burst. Burst
magnitude was larger in the neuron showing such an increase. B2: Summary histogram
illustrating the mean intraburst frequency and the mean peak frequency in neurons
showing (grey; n=25) or not showing (black; n=25) an increased firing rate prior to burst
30
occurrence. C: Correlation between burst magnitude (index) and firing rate prior to the
burst (mean frequency). Data were obtained from control experiments (black dots; n=39),
with 50-100 nM OT (grey triangles; n=5) and 100 nM d-OVT (empty squares; n=4). The
dotted line represents the linear regression through the data points obtained under control
conditions (r=0.61).
Figure 10: Contribution of OT-mediated rebound firing to OT neuron bursting activity.
A: histograms summarizing the percent change in the amplitude (q) and frequency
(Hz) of EPSPs (left panel) and IPSPs (right panel) in OT neurons showing (grey bars) or
not showing (black bars) an increased firing rate prior to burst occurrence. B: Sample
traces extracted from the recording shown in figure 9A at the time indicated. Detailed
analysis revealed a dramatic increase in IPSP activity and AP ( *) firing before burst
occurrence (b). C1: Superimposition of FHs before (grey) and after (black) application of
100 nM OT. Note that OT induced a marked increase in background activity and burst
magnitude. C2: FHs before (grey) and after (black) application of 100 nM d-OVT. Note that
the antagonist d-OVT decreased both background activity and burst magnitude. D:
Histograms summarizing the changes in background firing (mean Hz) and in burst
magnitude (index) caused by OT (grey) and d-OVT (black). The number of experiments
are indicated in brackets. E: Under OT treatment (grey bars), IPSPs occurring prior to the
burst showed a marked increase in their amplitude (q) and frequency (Hz), whereas dOVT (black bars) inhibited both parameters.